The invention relates to new macrophages, to a process for preparing the same and to their use as active substances of pharmaceutical compositions.
Cytotoxic leukocytes (NK, T lymphocytes, monocytes-macrophages) are potent effectors in the host defense against tumors. In vitro expansion and enhancement of the tumoricidal potential of autologous effector cells and their subsequent reinfusion into the host are currently being used in local or systemic adoptive immunotherapy (Rosenberg et al.: Progress report on the treatment of 157 patients with advanced cancer using LAK cells and IL2 or IL3 alone. N. Engl. J. Med. 316: 889, 1990; Rosenberg et al.: Use of tumor infiltrating lymphocytes and IL2 in the immunotherapy of patients with metastatic melanoma. N. Engl. J. Med. 319: 1676, 1990; Lacerna et al.: Adoptive cancer immunotherapy utilizing lymphokine activated killer cells and IFN-xcex3 activated killer lymphocytes. Pharm. Ther. 38: 453, 1988).
Macrophages play a central role in the antitumoral response and can be activated against neoplastic cells by immunopotentiators (Adams D. and Hamilton T.: Activation of macrophages for tumor cell kill: effector mechanism and regulation. In Heppner and Fulton (eds), Macrophages and cancer. CRC Press, 1988, p. 27; Fidler I.: Macrophages and metastases. A biological approach to cancer therapy. Cancer Res. 45: 4714, 1985).
In murine models, it has been demonstrated that activated macrophages given locally in the tumor inhibited tumor growth and decreased metastatic development (Bartholeyns et al.: Immunotherapy of cancer: experimental approach with activated macrophages proliferating in culture. Cancer Detect. and Prev. 12: 413, 1988; Chokri et al.: Antitumoral effect of LPS, TNF, IFN and activated macrophages: synergism and tissue distribution. Anticancer Res. 9: 1185, 1989). Human monocytes isolated from blood could be differentiated in vitro into macrophages by a 7 day culture in hydrophobic bags (Chokri et al.: Antitumoral effects of LPS, TNF, IFN, and activated macrophages: synergism and tissue distribution. Anticancer Res. 9: 1185, 1989). After incubation in the presence of IFN-xcex3, these macrophages became activated and tumoricidal, (Macrophages activated killer=MAK) for a number of human tumors in vitro or for tumors engrafted in nude mice (Andreesen et al.: Surface phenotype analysis of human monocytes to macrophages differentiation. J. Leuk. Biol. 47; 490, 1990; Dumont et al.: Control of the antitumoral activity of human macrophages produced in large amount in view of adoptive transfer. Eur. J. Cancer 24: 1691, 1988).
Adoptive transfer of MAK has undergone phase I clinical trials by different investigators in Strasbourg, Freiburg and Paris for patients with metastatic cancer infused systematically or intraperitoneally with 108 to 2xc3x97109 autologous MAK (Andreesen et al.: Adoptive transfer of tumor cytotoxic macrophages generated in vitro from circulating monocytes: a new approach to cancer immunotherapy. Cancer Res. 50: 7450, 1990; Bartholeyns et al. Adoptive immunotherapy of solid tumors with activated macrophages: experimental and clinical results. Anticancer Res. 11: 1201, 1991; Lopez et al.: One step separation by elutriation of monocytes from leukapheresis products of cancer patients for production of IFN-xcex3 activated macrophages in view of adoptive immunotherapy. J. Immunotherapy, 11: 209, 1992; Faradji et al.: Phase I trial of IV infusion of ex-vivo activated blood derived macrophages in patient with non small cell lung cancer: toxicity and immunomodulatory effects. Cancer Immunol. Immunother. 33: 319, 1991).
The clinical tolerance was excellent with minor side effects such as low grade fever and chills. The maximal tolerated dose could not be attained due to the limited number of MAK generated from one cytapheresis.
The recovery of larger quantities of MAK is therefore a need for optimal antitumoral efficacy in clinical trials and in therapy.
The macrophages prepared thus far in the conventional culture medium are obtained in a yield which is less than about 40%.
However, the need for macrophages presenting a higher cytotoxic activity is present in order to increase the efficacy in vivo and, subsequently, to delay the next macrophage administration.
One of the aims of the invention is to provide macrophages having a higher cytotoxic activity over standard macrophages.
Another aim of the invention is to provide macrophages in which the kinetics of the deactivation of the cytotoxic activity are slower with respect to standard macrophages.
Another aim of the invention is to provide a new culture medium which enables to prepare macrophages having an improved cytotoxic activity and slower deactivation kinetics of the cytotoxic activity with respect to standard macrophages.
The aims of the present invention are achieved by macrophages which have at least one of the following properties:
their cytotoxic activity without activation with IFN-xcex3 is increased by about 20 to 30% with respect to standard macrophages, and is preferably of about 70%;
their cytotoxic activity further to activation with IFN-xcex3 is increased by about 20 to about 40% with respect to standard macrophages, and is preferably of about 93%;
the extension of the deactivation time of the cytotoxic activity in reply to an activation of IFN-xcex3 is in a ratio such that after 60 h of activation with IFN-xcex3, the cytotoxic activity is higher than or equal to 30%, preferably of about 55%, compared to the maximum cytotoxic activity presented by the macrophages further to an activation with IFN-xcex3, with said cytotoxic activity being measured as the percentage of inhibition of 3-H thymidine incorporation by target tumoral cells, particularly U 937 cells.
The expression xe2x80x9cstandard macrophagesxe2x80x9d corresponds to the ones obtained by the culture of monocytes in a standard medium, such as defined in the Example section.
The xe2x80x9ccytotoxic activityxe2x80x9d corresponds to the one measured as the percentage of inhibition of 3-H thymidine incorporation by target tumoral cells, particularly U937 cells, and a test for this measure is hereafter given:
Differentiated macrophages were seeded into 96-well flat microtiter plates at 104 macrophages/well in 0.2 ml. After 2 h incubation at 37xc2x0 C. in 5% CO2 humidified atmosphere, the medium was removed and replaced by IMDM+5% hu AB serum containing or not 250 U/ml of rhu IFNxcex3; plates were further incubated overnight. After washing, 104 U937 tumor cells in 0.2 ml fresh medium (IMDM+10% FCS) were added to each well. After 24 h contact, 0.1 xcexcCi of tritiated thymidine was added to each well for a further 24 h incubation.
The cells were collected and the determination of incorporated radioactivity on the fiber glass filters was carried out by xcex2 counting. The percentage inhibition of thymidine incorporation by tumor cells could be calculated according to the formula:             (              cpmT        -        cpmTM            )        cpmT    xc3x97  100
where
cpmT=radioactivity in control tumor cells.
cpmTM=radioactivity in tumor cells+macrophages mixed culture.
It is to be noted that such method enables to determine the three main types of antitumoral activity of the macrophages described in the invention, i.e.: cytolysis, cytostasis and phagocytosis.
The macrophages originate from human subjects, who can be either healthy or patients suffering from various diseases.
By way of example, standard macrophages originating from culture of monocytes from healthy subjects present a spontaneous cytotoxic activity (i.e. without activation with IFN-xcex3) of about 45%, versus about 70%, in the case of the macrophages of the invention; and further to IFN-xcex3 activation, standard macrophages present a cytotoxic activity of about 76%, versus about 93%, in the case of the macrophages of the invention.
Similar enhancement of cytotoxic activity is obtained with macrophages issued from patients.
As to the kinetics of the deactivation time, table I hereafter gathers the results relative to the measure of the activity as described above in the case of standard macrophages and of macrophages of the invention.
From the above-mentioned properties, the advantages of the macrophages of the invention appear, knowing that cytotoxic activity below 50% is useless for therapeutics.
The macrophages of the invention are such that they make a weekly treatment possible, instead of a treatment every three or four days.
According to a preferred embodiment, the macrophages of the invention have the following characteristics:
their size is from about 10 to about 20 xcexcm;
they adhere to plastic surface;
their viability is preferably higher than about 70%;
they present a phagocytosis property;
they present on their surface antigens of differentiation, such as CD64, CD68, MAX1, HLADR;
their cytotoxic activity is higher than about 50%;
the macrophages of the invention are advantageously free of bacterial and biological contaminants.
When the size of the macrophages is higher than 20 xcexcm, it means that they tend to fuse between one another to form giant cells, the role of which is not clearly understood; when the size is smaller than 10 xcexcm, it means that the macrophages are not well differentiated.
The size can be measured with a channelyser (Coulter Margency-France)
The adherence property to plastic surface can be verified on Petri dishes (% adherence) after 30 minutes of incubation of the produced macrophages.
The viability is the number of alive cells at the end of the culture, and the viability is preferably higher than about 70% and is measured by the Trypan Blue exclusion.
The phagocytosis property can be checked by ingestion of latex beads or dextran particles (30 minutes to 1 h of incubation), as described by Stevenson and Fauci, Manual of Macrophages methodology; Marcel Dekker,. N.Y. pp. 75-80, 1981.
The surface antigens of differentiation such as CD64, CD68, MAX 1, HLADR are expressed at a similar or higher level with respect to standard macrophages; this can be measured by flow cytometry. Table II hereafter gives the results of phenotypes obtained under different conditions.
From this table, it can be concluded that the macrophages of the invention have new properties but the main functions of macrophages have not been altered, with the exception of the increase of the cytotoxic activity.
However, the CD71 which is the transferrin receptor is decreased in the presence of VitD3+GM-CSF which correlates with an advanced activation status of the macrophages. The CD71 expression is determined as described in the Example section.
The cytotoxic activity is determined with U937 cells, the ratio between macrophages and U937 being of 1/1.
The macrophages of the invention are advantageously devoid of viral, bacterial, fungal and biological contaminants.
The invention also relates to a process for preparing macrophages as defined above, comprising the culture of monocytes in a culture medium containing 1,25-dihydroxy D3 vitamin and GM-CSF.
More particularly, the process of the invention comprises the culture of both monocytes and lymphocytes in a culture medium containing 1,25-dihydroxy vitamin D3 and GM-CSF for a time sufficient to obtain differentiated macrophages, preferably for about 6 or 7 days, possibly activating the macrophages resulting from the monocytes and lymphocytes with IFN-xcex3, and separating the macrophages from the lymphocytes, before or after the activation with IFN-xcex3, preferably after IFN-xcex3 activation, and recovering the macrophages.
According to a preferred embodiment, the process of the invention comprises the following steps:
isolation of leukocytes from blood of healthy subjects or cancerous patients by cytapheresis, to obtain the cytapheresis products (i.e. concentrated leukocytes),
platelet elimination, for instance by centrifugation of the cytapheresis products, to obtain a leukocyte enriched product,
separation, in the leukocyte enriched products, of the mononuclear cells on one hand, and of the contaminating red blood cells and granulocytes on the other hand,
culture of the mononuclear cells (monocytes+lymphocytes) in a medium containing 1,25-dihydroxy vitamin D3 and GM-CSF for about 6 or 7 days, to obtain differentiated macrophages,
activation of the macrophages and lymphocytes with IFN-xcex3 for about 16 h to about 24 h,
separation of the activated macrophages from the lymphocytes for instance by elutriation.
The lymphocytes can be separated from the monocytes before the culture step.
The lymphocytes can be separated from the macrophages after the culture and before the IFN-xcex3 activation. Advantageously the macrophages and lymphocytes are separated from each other after IFN-xcex3 activation.
In the process of the invention, D3 vitamin is used at a concentration of 10xe2x88x9210 to about 10xe2x88x927 preferably of about 10xe2x88x928 M.
In the process of the invention, GM-CSF is used at a concentration of about 50 to about 1000 U/ml, particularly of about 100 to about 500 U/ml.
In the process of the invention, the culture medium is RPMI, IMDM, MEM, or DMEM.
These media are commercially available.
Advantageously, the culture medium contains indomethacin (or another cyclo-oxygenase inhibitor) or/and cimetidine (an histamine H2 antogonist).
An advantageous process for preparting the macrophages of the invention is the following:
Cytapheresis
Leukocytes from healthy subjects or from cancerous patients are isolated from peripheral blood by cytapheresis using either the Cobe 2997 or the Dideco Vivacell continous-flow blood cell separators. The cytapheresis product is centrifuged for 10 min at 280 g in order to reduce platelet contamination. The platelet-enriched plasma is removed and leukocyte pellet resuspended in a phosphate buffer solution (PBS) containing 0.1% glucose, 0.17% PO3HNa2, 2H2O, 0.27% PO3H2Na, 0.14% NH4Cl, 0.78% NaCl (solution TS745 laboratoire Bruneau, France).
The enriched leukocyte pellet is obtained with an average of 7 to 9xc3x97109 leukocytes (50% of mononuclear cells).
Isolation of Mononuclear Cells
Human mononuclear cells are separated from red blood cells and from contaminating granulocytes, by 15 min centrifugation at 1000 g on a COBE 2991 or Stericell cell processor using Ficoll Paque of density 1.077 (Pharmacia). After 3 washings in phosphate buffered saline solution without calcium and magnesium, the monocytes are obtained with about 50% purity as shown by channelyser analysis (Coulter Margency-France).
Culture
Differentiated human macrophages are obtained by 7 days in culture of monoclear cells in hydrophobic bags in Teflon or polypropylene (Dupont-J. BIO, life cell-Travenol stericell-TERUMO) at 37xc2x0 C. and 5% CO2, 95% humidified atmosphere. Total mononuclear cells are seeded at 5.106 cells/ml in Iscove modified medium (IMDM, Gibco) or equivalent medium supplemented by penicillin (100 UI/ml), streptomycine (100 xcexcg/ml), L-glutamine (2 mM, Gibco), pyruvic acid (2 mM, Gibco), Indomethacin (5.10xe2x88x926M, Sigma), cimetidine (10xe2x88x926 to 10xe2x88x929M), mercaptoethanol (3.10xe2x88x925 M, Gibco) non essential amino-acids (1%, Gibco) and 2-5% of autologous or AB serum. The addition of GM-CSF (500 U/ml, Shering) and/or 1,25 dihydroxy-Vit D3 (cholecalciferol 10xe2x88x928M, Roche, Basel, Sw) was done in comparative experiment.
Activation of macrophage to cytotoxicity was performed by a further 16-18 h culture in presence of 250 U/ml of IFN-xcex3 (Boehringer, Ingelheim, FRG).
Elutriation
The differentiated macrophages obtained after 7 days of culture and after activation are centrifuged at 550 g for 10 min and the pellet is resuspended in phosphate buffered saline solution containing 0.1% glucose, 0.17% PO3HNa2, 2H2O, 0.27% PO3H2Na, 0.14% NH4Cl, 0.78% NaCl (solution TS745 laboratoire Bruneau, France) with 2% human serum albumin (HSA, CNTS-France).
The cells are then subjected to elutriation using a Beckman J6 ME centrifuge equipped with a J5.O rotor and a 40 ml elutriation chamber as described by Andreesen et al. (Cancer Res., 50, 1990).
The differentiated macrophages are collected at a constant rotor speed and increasing the flow rate.
The process of the invention enables to obtain a yield of macrophages higher than about 40%, and preferably of about 76%, with said yield being the ratio between the obtained live macrophages and the starting monocytes. This yield can be determined by cell counting and channelyser.
According to a preferred embodiment, the process of the invention is such that killed tumoral cells are added into the culture medium simultaneously with monocytes, both cells coming preferably from the same patient, preferably at the ratio of about 1 million of killed tumoral cells/ml, with said killed tumoral cells being processed at the same time as macrophages.
The killed tumoral cells can then be processed simultaneously with the leukocytes, in an amount of about 1xc3x97106/ml, and can be separated from the macrophages for instance at the same time as the lymphocytes, after IFN-xcex3 activation or before IFN-xcex3 activation, and preferably after IFN-xcex3 activation.
This process allows to obtain macrophages and lymphocytes specific for the tumor, killing very efficiently in vivo these specific tumor cells.
The invention also relates to the macrophages liable to be obtained according to the above-defined process.
The invention also relates to pharmaceutical compositions containing, as active substance, macrophages as defined above.
The invention also relates to a medium containing elements necessary for the growth and differentiation of monocytes into macrophages of the invention, and in addition containing D3 vitamin and GM-CSF.
The macrophages of the invention can be part of a kit containing:
means for the recovery of lymphocytes and monocytes free of contaminants;
appropriate buffer and wash solutions, and possibly appropriate means for the conservation of macrophages;
means for preparing a culture medium for the monocytes and possibly the lymphocytes and containing 1,25-dihydroxy vitamin D3 and GM-CSF;
possibly IFN-xcex3.
According to an advantageous embodiment of the invention, the kit contains:
means for recovering and centrifuging blood to obtain a leukocyte concentrate;
means for separating lymphocytes and monocytes from the other white cells and for eliminating the contaminating red cells;
culture medium for macrophages and possibly lymphocytes with complements and particularly 1,25-dihydroxy vitamin D3 and GM-CSF and possibly indomethacin and/or cimetidine;
means for the separation of lymphocytes from macrophages;
appropriate means for the conservation of macrophages;
appropriate buffer and wash solutions;
possibly IFN-xcex3.
By way of examples, an advantageous kit comprises the following elements:
1xe2x80x94Cytapheresis kit (Fenwal),
2xe2x80x94Transfer bag (R 20-21-Fenwal),
3xe2x80x94Kit of centrifugation under Ficoll gradient (Gradient/wash processor set Stericell-Terumo) or blood cell processor (Cobe 912-647-819),
4xe2x80x94Elutriation Kit (Macopharma),
5xe2x80x94Transfert Pack Unit 2L (Travenol-4R2041),
6xe2x80x94Hydrophobic bag for cell culture (Teflon, J. BIO-lifecell, Travenol-Stericell, Terumo),
7xe2x80x94Injection bag (Travenol),
8xe2x80x94Phosphate buffered saline solution (solution TS745 Laboratoires Bruneau),
9xe2x80x94Ficoll-Paque (Pharmacia),
10xe2x80x94Human Albumin solution (Biotransfusion),
11xe2x80x94Culture medium (IMDM-Gibco),
12xe2x80x94Penicillin/Streptomycine (Gibco),
13xe2x80x94L-Glutamine (Gibco),
14xe2x80x94Non essential amino-acids (Gibco),
15xe2x80x94Pyruvic acid (Gibco),
16xe2x80x94Indomethacin (Sigma),
17xe2x80x94Mercaptoethanol (Gibco),
18xe2x80x94GM-CSF (Shering),
19xe2x80x941,25 dihydroxy-Vit D3 (Roche, Basel, Sw),
20xe2x80x94Vial of IFN-xcex3 (Boehringer).
The invention also relates to products containing macrophages according to the invention, and lymphocytes, as a combined preparation for simultaneous, separate or sequential use in adoptive immunotherapy.
According to an advantageous embodiment, the products of the invention as defined above are characterised in that they contain the macrophages and the lymphocytes in a ratio of at least ⅓-⅔expressed in cell number.
In this embodiment, the macrophages and the lymphocytes are both injected to a patient. This can mean that:
either the elutriation step is cancelled and the macrophages and lymphocytes are injected
after IFN-xcex3 activation;
or after simultaneous IFN-xcex3 and IL-2 activation;
or the macrophages are separated from each other after the culture, but before activation, with macrophages being subsequently activated with IFN-xcex3 and lymphocytes being subsequently activated with IL-2.
The invention also relates to bispecific antibodies liable to recognize an antigen of a macrophage of the invention and an antigen of a tumoral cell which is to be targetted by said macrophage.
The bispecific antibodies can be prepared as described in Chokri et al. Res. Immunol. 143 (1992).
The bispecific antibodies can be injected at the same time as the macrophages of the invention, or can be pre-incubated with macrophages before injection.
The invention also relates to a method for the treatment of cancer, comprising the administration of an appropriate amount of macrophages according to the invention, and preferably in an amount of about 2xc3x97109 to about 5xc3x97109 macrophages.
A method according to the invention for the treatment of cancer comprises the administration of lymphocytes in an amount of about 4xc3x97109 to about 10xc3x97109 lymphocytes.
The macrophages of the invention can also be used as nucleic acid and/or drug vectors. The invention thus comprises macrophages containing exogenous nucleic acids and/or drugs.
Regarding mnacrophages containing exogenous nucleic acids, the macrophages are prepared. By transfection, DNA coding for a cytokine or for a defective protein, or for a tumor antigen, is integrated into the macrophages and it is checked that it is expressed on the macrophages before injecting it into the patient.
The long survival of the macrophages allows the in vivo expression of the integrated protein genes.